Avian, vitelline antibodies directed against HIV antigens

ABSTRACT

The present invention is drawn to avian vitellin antibodies that are immunologically reactive with HIV antigens, a process for the preparation thereof, a use thereof and a food substance in the form of a hen&#39;s egg containing the antibodies. The antibodies of the present invention are produced by immunizing hens with HIV antigens, in particular HIV core antigens and concentrating the antibodies in the egg yolk. The antibodies are suitable for passive immunization of mammals.

The invention relates to avian, vitelline antibodies directed againstHIV antigens, methods for their preparation, and their use. Theseantibodies—monospecific, polyclonal egg yolk antibodies from SPFchickens (specified pathogen-free chicken)—are suitable in HIV (humanimmunodeficiency viruses) diagnostics and in the therapeutical use onHIV-positive patients in the symptom-free stage, and where symptoms ofARC (AIDS-related complex) or AIDS (acquired immunodeficiency syndrome)are present.

Since the end of the last century it is well-known that considerableamounts of antibodies (Ab) may be accumulated in chicken egg yolk(Klemperer F., Arch. exptl. Pathol. Pharmakol. 31 (1893) 356-382). Overthe last ten years, there were massively increasing efforts to recoverantibodies from bird eggs and utilize them for medical purposes. Incontrast to other methods, the antibody accumulation and recovery viabird's egg is a bloodless method, since blood withdrawal is not requiredfor Ab recovery (Schade, R. and A Hlinak: Mh. Vet.-Med. 48 (1993) 91-98,Gustav Fischer Verlag, Jena).

The well-known methods comprise immunization of chickens with an antigenand recovery of the antibodies (IgY, yolk antibodies) from the eggs/eggyolk (US=US patent, GB=British patent, DD=East German application,DE=German unexamined application (OS)/patent specification (PS),EP=European patent, WO=PCT(Patent Cooperation Treaty) application:Immunologically reactive preparations (Egg Yolk Antibodies, Polson, A.:DE 29 51 412), Egg Yolk Antibodies (Polson, A.: GB 2,057,451, U.S. Pat.No. 4,357,272), Fowl Egg Antibodies (Polson, A.: U.S. Pat. No.4,550,019), Specific Chicken Egg Antibodies (Tsuda, K. et al.: EP503,293), Specific antibody-containing substance from eggs (Tokoro, H.:U.S. Pat. No. 5,080,895, EP 225,254).

Passive immunization of mammals using antibodies recovered from a fowlspecies has also been described (Stolle, R. and L. R. Beck: DE 35 04221, U.S. Pat. No. 4,748,018, EP 152,270). Therein, immunizingquantities of an antibody recovered from eggs of a fowl speciesimmunized with the antigen causing the mammal's disease are administeredto the mammal.

Another well-known example is the successful passive immunizationagainst rotavirus infections in mammals using avian, vitellineantibodies (Bartz, C. R. et al., The Journal of Infectious Diseases,Vol. 142, No. 3, 1980, 439-441; Yolken, R. H. et al., Pediatrics (1988)81 (2) 291-295; Kuroki, M. et al., Veterinary Microbiology, 37 (1993)135-146); and the passive immunization of mammals using avian antibodiesagainst E. coli (Wiedemann, V. et al., Journal of Veterinary Medicine,Series B (1990) 37 (3), 163-172; ibid., (1991) 38(4), 283-291; Yokoyama,H. et al., Infection and Immunity (1992) 60 (3), 998-1007).

The structural differences between avian and mammal IgG are explained onthe basis of the phylogenetic divergence of birds and mammals. Aboveall, they are apparent from the differences in molecular weights andsedimentation constants: chicken IgG 170,000 or 174,000, human IgG150,000 daltons (Larsson, A. and J. Sjöquist, Comp. Immun. Microbiol.infect. Dis., Vol. 13, No. 4, pp 199-201, 1990; Jürgens, L.: Reinigungvon IgG und IgG-Antikörpern aus dem Eidotter, Ph.D. Thesis 1987,Ludwig-Maximilians-Universität, Munich).

It is also well-known that HIV-infected cells express core antigens ontheir cell membranes, e.g. p24 antigen (Nishino, Y. et al., Vaccine,1992, 10 (10) 677-683). An in vitro inhibition of HIV infection usinganti-p24 antibodies is described by Gregersen J. P. et al. (J. Med.Virol. 1990, 30(4) (287-293) and Franke, L. et al. (J. Med. Virol. 1992,37 (2) 137-142). The influence of a high anti-HIV p24 antibody titer onthe condition of HIV-positive patients is the subject matter of WO89/01339 (Cummins, L. M. et al.), and that with the proteins gp41 andp24 of corresponding antibodies is the subject matter of WO 90/09805(Zolla-Pazner, S. et al.). The assessment of the immunologic defensepattern of a patient is the essence of DD 299 090, according to whichthe anti-HIV Ab content of the patient after treatment with monoclonalantibodies (mAb) particularly directed against the HIV protein p24(anti-HIV p24 mAb) is determined using ELISA (Enzyme-LinkedImmunosorbent Assay).

The symptoms of ARC and AIDS, respectively, begin with a massivedecrease or total disappearance of the anti-p24 antibodies from theserum of HIV-positive patients (Jackson, G. G., The Lancet, Sep. 17,1988, 647-651; Karpas, A. et al., Proc. Natl. Acad. Sci. USA, Vol. 87,pp 7613-7617, October 1990), whereas patients having a high anti-p24antibody titer remain symptom-free. Several authors conducted a passiveimmunization with patients in the ARC and AIDS stages (Karpas, A. et al.1990, ibid.; Karpas, A. et al., Int. Conf. AIDS, Jun. 6-11, 1993 9 (1),244, Abstract No. PO-A28-0659; Jackson, G. G., ibid.; Hague, R. et al.,Int. Conf. AIDS, Jun. 4-9, 1989 , 5, 328, Abstract No. T.B.P. 246;Lefrere, J. J. et al., a) Int. Conf. AIDS, Jun. 6-11, 1993 9 (1), 246,Abstract No. PO-A28-0667; b) Rev. Fr. Transfus. Hemobiol., May, 34 1994(3), 199-211; c) Int. Conf. AIDS, Aug. 7-12, 1994 10 (1), 226, AbstractNo. PBO335), where plasma from symptom-free patients (up to 500 ml) withhigh anti-p24 antibody titer was transfused to patients in the ARC andAIDS stages after the viruses in the plasma had been inactivated byheat. Two hours after transfusion, p24 could not be detected in theserum anymore; also, the number of HIV-infected cells was reduced.Patients in the ARC stage showed significant amelioration of theclinical symptoms up to complete absence of symptoms. There was nodecrease in the number of T4-helper cells anymore.

To date, survival with absence of symptoms for periods as long as 22 and35 months has been described. With patients in an advanced stage ofAIDS, remission continued for 6 to 22 months, depending on the state ofthe individual severity level of the disease at the time treatment hadbegun, with present aviremia and sufficient antibody level. In the ARCstage, the clinical remission continued 22 months beyond the KARPASstudy, with present aviremia and stable CD4+ T-cell number (Karpas, A.et al., Int. Conf. AIDS, Jun. 6-11, 1993, 9 (1), 244, Abstract No.PO-A28-0659; Bainbridge, D. et al., Int. Conf. AIDS, Aug. 7-12, 1994, 10(1), 216, Abstract No. PB 0293).

The invention is based on the object of providing antibodies directedagainst HIV antigens, which may be administered to HIV-infectedpatients. The object was accomplished by immunizing chickens with HIVantigens, thereby accumulating anti-HIV antibodies in the egg yolk.Surprisingly, it was found that administering these antibodies in theform of an oral administration of dried egg yolk or as antibody extractafter isolation from egg yolk and work-up results in increased antibodyserum titer values, and that avian antibodies in mammals are capable ofbinding the mammal complement.

As a result of the phylogenetic divergence between birds and mammals,fowl species for the production of antibodies against mammal diseaseshad been excluded, particularly for the reason that chicken protein wasforeign to the human immune system and would give rise to allergicreactions when used repeatedly (DE 35 04 221 C2 dated May 19, 1994).According to Gippner-Steppert et al., chicken antibodies —in contrast tomammal antibodies—would give no reaction with mammal complement factorC1 and mammal Fc-receptors (Gippner-Steppert, C. and M. Jochum:“Production and purification of chicken polyclonal anti-peptideantibodies specific for fibrino-elastase-peptide A-alphal-21”, lectureheld during the public annual colloquium of the Surgical-Medical Clinicof the LMU Munich, Feb. 4, 1994).

The antibodies of the invention are novel both as avian antibodies andin their structure. They differ from well-known antibodies in molecularweight, sedimentation constant and isoelectric point.

According to the invention, particularly those antibodies are used whichare directed against HIV core antigens. The most important role isassigned to the antibody derived from the core antigen p24, followed bythe HIV proteins p17, p7 (9), p12, p66, and p32 (Kageyama, S. et al.,Int. Conf. AIDS, Aug. 7-12, 1994, 10 (2), 86, Abstract No. PA 0224).

According to present knowledge, it appears that the anti-p24 antibodiesof HIV-positive patients (HIV-1 and HIV-2) protect most effectively fromthe ARC and AIDS stages to occur. It is not clear as yet, to what extentother antibodies have an additional effect against core antigens.

The antibody titer against core protein p17 decreases earlier than theanti-p24 antibody (Lange, J. M. et al., AIDS, Sep. 1 1987, (3), 155-59).

The anti-core antibodies of the invention, and particularly the antibodyagainst p24 antigen, result in absence of symptoms to a large extent andprolongation of life in ARC stage patients.

For mere logistic reasons, however, only a small circle of affectedpersons may benefit from the plasma transfusion method of treatmentdescribed above.

The avian, vitelline antibodies against core proteins according to theinvention offer the advantage that they are produced in large amounts bychickens and transferred to the egg yolk. According to the invention,the antibodies are administered parenterally in a highly purified form,but also on the oral route as dry egg yolk or antibody extract, becausethey are thermally stable and acid-resistant to a high extent. With thesolution of the invention, a new kind of food is offered: the chicken'segg containing anti-HIV antibodies, wherein the antibodies areaccumulated in the egg yolk in particular, but not exclusively.

In particular, core antigens, preferably p24 and p17 are suitable as HIVantigens. Using SPF chickens, they result in the corresponding SPFchicken polyclonal antibodies according to the invention.

The method of the invention for producing avian, vitelline antibodiesdirected against HIV antigens comprises immunization of chickens withHIV antibodies, particularly core antigens, preferably the core proteinsp24 and/or p17, thereby accumulating the antibodies in the egg and eggyolk, respectively, and optional isolation therefrom.

The antibodies of the invention may be used in the passive immunizationof mammals. They are suited to be administered to HIV-positive patientsin order to produce high antibody serum titers.

The isolation of the IgY antibodies of the invention is effected fromaqueous yolk solutions according to well-known procedures, using PBS(phosphate-buffered sodium chloride solution) as solvent and subsequentcentrifugation, precipitation of immunoglobulins by means ofprecipitating agents such as polyethylene glycol (PEG), sodium, dextranor ammonium sulfate and/or chromatographic purification such as gelfiltration or ion exchange (Schade, R. and A. Hlinak: Mh. Vet.-Med. 48,1993, p. 95, Gustav Fischer Verlag, Jena).

With reference to the embodiments, the invention will be illustrated inmore detail.

Embodiments EXAMPLE 1

General procedure

The immunization of chickens, especially SPF chickens, is effected usingsynthetic, non-infectious HIV core complete antigens and

a) Freund's complete adjuvant (FCA)

b) Freund's incomplete adjuvant (ICFA)

The sampling of blood and egg yolk was effected using pools eachcomprising 5 chickens of the antigen test group and a negative controlgroup. The injection dose was 1.0 ml i.m. into the pectoral muscles into4 depots each having 0.5 ml of resuspended synthetic antigen andadjuvant.

Start: Young chickens from the 15th week of life on.

End: 70th week of life (end of laying).

1.1. Initial immunization (base immunization) using 200 μg of syntheticHIV core complete antigen (protein) and FCA and blood zero sample on day“0” (I₀ sample).

1.2. 1st boosting (2nd immunization) on day 28 with blood and egg yolksample I₁ for serological antibody control (IgG/IgY), including negativecontrol group. All boostings were carried out using ICFA.

1.3. 2nd boosting (3rd immunization) on day 56 with I₂ blood and eggyolk samples including negative control group.

1.4. 3rd boosting (4th immunization) on day 84 with I₃ blood and eggyolk samples including negative control group.

1.5. I₄ blood and egg yolk samples on day 98, including negative controlgroup.

1.6. I₅ blood and egg yolk samples on day 112, including negativecontrol group.

1.7. I₆ blood and egg yolk samples on day 140, including negativecontrol groups.

1.8. I₇ blood and egg yolk samples on day 168, including negativecontrol groups.

1.9. 4th boosting (5th immunization) on day 220 with I₈ blood and eggyolk samples including negative control groups.

1.10. Egg yolk isolation and yolk separation are effected according to abatch process including final purification and extraction of thespecific HIV antibodies against core complete antigens (proteins)(Schade R. and A. Hlinak, Mh. Vet.-Med. 48, 1993, p. 95, Gustav FischerVerlag, Jena: IgY- Präparation, Extraktion vitelliner Antikörper).

1.11. IgY determination

The qualitative and quantitative determinations of the specificpolyclonal IgY egg yolk antibodies amounting to 175 mg of antibodies/eggyolk are effected according to Schade R. and A Hilinak (1993), ibid.

The quantitative determination is effected via specific antibody titerexaminations on yolk homogenizate using indirect ELISA, and that of thespecific antibody content/quantity unit of yolk is effected via affinitychromatography (using purified and separated antibody extract aftervacuum dialysis).

EXAMPLE 2

Use of HIV p24 Antigen

As in Example 1, using HIV core p24 antigen.

EXAMPLE 3

Use of HIV p17 Antigen

As in Example 1, using HIV core p17 antigen.

EXAMPLE 4

Isolation of Pure Antibodies

This is effected according to a batch process (Schade R. 1993, cf.,Example 1.10), for parenteral and oral therapy.

What is claimed is:
 1. Avian, vitelline antibodies which areimmunologically reactive with HIV antigens.
 2. The antibodies accordingto claim 1, wherein said antibodies are immunologically reactive withHIV core proteins.
 3. The antibodies according to claim 1 or 2 areimmunologically reactive with HIV core proteins p24 and p17.
 4. Avian,vitelline polyclonal antibodies directed against HIV antigens, whereinsaid antibodies are produced by immunizing chickens with HIV antigensand preparing the egg yolk from eggs laid by the chickens.
 5. Theantibodies according to claim 4, wherein said HIV antigens are HIV coreproteins, and SPF (specified pathogen-free) chickens are immunized withsaid HIV core proteins.
 6. The antibodies according to claim 5, whereinsaid core proteins are core proteins p24 and/or p17.
 7. A method ofpassive immunization comprising administering the antibodies accordingto claim 1 or 4, to a mammal.
 8. A method for producing avian, vitellineantibodies immunologically reactive with HIV antigens, which comprisesimmunizing chickens with HIV antigens, and accumulating the antibodiesin the egg yolk.
 9. The method according to claim 8, wherein HIV coreantigens, are used as antigens.
 10. A method for producing high antibodyserum titers against HIV antigens which comprises parenterallyadministering to an HIV-positive patient in need thereof avian,vitelline antibodies directed against HIV antigens.
 11. The method ofclaim 9, wherein said HIV core antigens are core proteins p24 and/orp17.